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Mesenchymal stem cell (MSCs) therapy protects donor lungs against ischemic-reperfusion injury. We recently reported that local infusion of human umbilical cord-derived MSCs during donor lung ablation significantly conserved lung function. The anti-inflammatory effect of MSCs is mainly exerted through a paracrine mechanism. Thus, in the present study, we investigated whether the preservation capacity of MSCs is related to the secretion of anti-inflammatory exosomes. The rat lung preservation model settled before consisted on: cardiac arrest, warm ischemia (2h) in which cells were administered at 1h via the pulmonary artery (1 × 10^6 cells or vehicle), cold ischemia, and normothermic ex-vivo lung perfusion with ventilation. Here, we assessed 3 experimental groups right after the warm ischemia period (2h): vehicle, MSCs, and exosomes (released by 1 × 10^6 cells). Exosomes were chromatographically purified from MSCs conditioned medium and their anti-inflammatory activity was evaluated, in vitro, based on their ability to suppress the acquisition of M1 phenotype in LPS-stimulated RAW264.7 cells. Physiologic data (pressure-volume curves) were acquired right after the cardiac arrest and at the end of the warm ischemia time and lung biopsies were taken to study edema formation, neutrophil infiltration, redox profile, and inflammatory molecular markers. As expected, exosomes were positive for human-specific CD63 and CD81, and showed the tipical size and morphology by electron microscopy. Exosomes also exposed their anti-inflammatory activity by significantly reducing IL-6 secretion in LPS-stimulated macrophages by 60%. Previously, in the complete lung preservation model (4.5h), we observed that MSCs administration facilitated organ preservation by significantly reducing the drop on lung compliance (∼50% vs vehicle treated group, p<0.001) and controlling reactive oxygen species production. Interestingly, after 2h of warm ischemia, lung compliance dropped by 42% (p<0.001) in the exosomes-treated group, while the vehicle and MSCs groups showed a similar and much stronger reduction (73% and 76%, respectively). Histologic assessment, tissue oxidative profile determination and molecular analysis are ongoing. Taking in consideration our previous data and these new results we conclude that MSCs-derived exosomes have a key role in lung preservation mediated by MSCs. Consequently, we postulate that exosomes may represent an innovative cell free therapy for organ preservation. [ABSTRACT FROM AUTHOR]

Recently, we reported that local infusion of human umbilical cord-derived mesenchymal stem cells (MSCs) during donor lung ablation may increase lung availability by improving organ preservation. We found that MSCs protect donor lungs against ischemic injury by reducing oxidative damage and neutrophil extravasation. Pre-conditioning has emerged as a strategy to improve MSC-based therapies. Here, we studied whether the preservation capacity of MSCs may be enhanced by their pre-activation into an anti-inflammatory phenotype with TNF-alpha (TNF-a). We settled a lung preservation model in rats that mimics the different phases by which donor organs must undergo before implantation which consist on: cardiac arrest, warm ischemia (2h at room temperature), cold ischemia (1.5h at 4°C, with Perfadex), and normothermic lung perfusion with ventilation (Steen solution, 1h). After 1h of warm ischemia, 1 × 10^6 cells (MSCs, or TNF-a treated cells) or vehicle were infused via the pulmonary artery. Physiologic data (pressure-volume curves) were acquired right after the cardiac arrest and at the end of the perfusion. Lung biopsies were taken at the end of the perfusion to study edema formation, neutrophil infiltration, redox profile, etc. Interestingly, although the TNF-a treated cells showed an anti-inflammatory phenotype in vitro, we did not register a significant change in their preservation capacity. In this line, lung compliance decreased 39% and 35% in the MSCs- and TNF-a-treated groups, respectively, while the vehicle group showed a stronger reduction (72%, p<0.001). Histologic assessment showed the presence of human cells in the microvasculature of the cell-treated lungs. Consequently, the cell therapy groups presented a lower injury score (p<0.01), and a significant reduction of the MPO activity (a neutrophil marker) compared with vehicle (p<0.001). Cell-treated lungs significantly decreased tisular oxidative damage by controlling the reactive-oxygen species production. Catalase and superoxide dismutase enzyme activities were increased in the vehicle group. These results suggest that, in this therapeutic conditions, the anti-inflammatory effect of non-activated MSCs is strong enough to conserve the functionality of the lung during ischemia, and reaffirm the notion that cell therapy is a novel tool for organ preservation. [ABSTRACT FROM AUTHOR]

Exosomes (Exo) are small sized-extracellular vesicles (40- 150 nm), released by almost all kind of cells, and play a major role in cell-to-cell communication. In the last years, they have drawn attention due to its potential application in clinical diagnostics and therapeutics. However, determining their biological activity and potency has proven difficult, owing to the lack of biological assays. Here, we standardized an in vitro assay to assess the antiinflammatory potential of mesenchymal stem cells (MSCs)-derived exosomes based on their ability to prevent the acquisition of the M1 phenotype in LPS-stimulated RAW264.7 macrophages. M1 phenotype was characterized by the induction of IL-1β, IL-6 and iNOS as determined using qRT-PCR. Nitric oxide (NO) released by iNOS turns into NO2-, that can be easily quantitated in the culture media by Griess reaction. Moreover, phenol red present in culture media did not interfere in the spectrophotometric detection of NO2-. Thus, we first tested different assay conditions in 96-well-plates, including two seeding densities (2 × 104 and 4 × 104 cells), four LPS doses (1, 10, 100 and 1000 ng/ml) and two timepoints (16 and 24 h), in order to determine the best set-up to accurately measure NO2- production as a marker of M1 macrophage polarization. We found that seeding 2 × 104 cells/well and stimulating with 10 ng/ml LPS for 16 h allowed us to inhibit the inflammatory response by 60% using Dexamethasone (1 ug/ml). Using these established conditions, we were able to test different exosomes preparations in sextuplicate (5 μg protein/well) and to rank then by their anti-inflammatory activity. Finally, conditioned media containing NO2- can be processed immediately or stored at -20°C, as we found that NO2- is stable in culture media. In summary, we standardized a quick, cheap and reproducible in vitro macrophage assay that allows evaluating and estimating the antiinflammatory activity of MSCs-derived exosomes. The assay is convenient for comparing multiple samples and, therefore, should be useful in developing protocols to improve the purification and characterization of antiinflammatory exosomes. [ABSTRACT FROM AUTHOR]